TGFbeta type II-type III receptor fusions

ABSTRACT

Certain embodiments are directed to novel heterotrimeric fusions in which the ectodomain of the TGF-β type II receptor (TβP?II) is coupled to the N- and C-terminal ends of the endoglin-domain of the TGF-β type III receptor (TpRIIIE). Certain embodiments are directed to novel heterotrimeric polypeptides in which the ectodomain of the TGF-β type II receptor (TI3RII) is coupled to the N- and C-terminal ends of the endoglin-domain (E domain) of the TGF-β type III receptor (TI3RIII). This trimeric receptor, known as RER, can bind all three TGF-β isoforms with sub-nanomolar affinity and is effective at neutralizing signaling induced by all three TGF-β isoforms, but not other ligands of the TGF-β superfamily, such as activins, growth and differentiation factors (GDFs), and bone morphonogenetic proteins (BMPs).

PRIORITY PARAGRAPH

This application is a U.S. National Stage Application of InternationalApplication serial number PCT/US2013/034504 filed Mar. 28, 2013, whichclaims priority to U.S. Provisional Application serial number 61/616,740filed Mar. 28, 2012. This application claims priority to andincorporates by reference each of the above referenced applications intheir entirety.

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

This invention was made with government support under CA079683 andGM58670 awarded by the National Institutes of Health. The government hascertain rights in the invention.

REFERENCE TO SEQUENCE LISTING

A sequence listing is being submitted electronically with thisapplication. The sequence listing is incorporated herein by reference.

BACKGROUND

Transforming growth factor beta (TGFβ) isoforms (β1, β2, and β3) arehomodimeric polypeptides of 25 kDa. They are secreted in a latent formand only a small percentage of total secreted TGFβs are activated underphysiological conditions. TGFβ binds to three different cell surfacereceptors called type I (RI), type II (RII), and type III (RIII)receptors. RI and RII are serine/threonine kinase receptors. RIII (alsocalled betaglycan) has two TGFβ binding sites in its extracellulardomain, which are called the E and U domains (BG_(E) and BG_(U),respectively). TGFβ1 and TGFβ3 bind RII with an affinity that is 200-300fold higher than TGF-β2 (Baardsnes et al., Biochemistry, 48, 2146-55,2009); accordingly, cells deficient in RIII are 200- to 300-fold lessresponsive to equivalent concentrations of TGF-β2 compared to TGF-β1 andTGFβ-3 (Chiefetz, et al (1990) J. Bio. Chem., 265, 20533-20538).However, in the presence of RIII, cells respond roughly equally to allthree TGF-β isoforms, consistent with reports that show that RIII cansequester and present the ligand to RII to augment TGFβ activity when itis membrane-bound (Chen et al., J. Biol. Chem. 272, 12862-12867, 1997;Lopez-Casillas et al., Cell 73, 1435-1444, 1993; Wang et al., Cell 67,797-805, 1991; Fukushima et al., J. Biol. Chem. 268, 22710-22715, 1993;Lopez-Casillas et al., J. Cell Biol. 124, 557-568, 1994). Binding ofTGFβ to RII recruits and activates RI through phosphorylation (Wrana etal., Nature 370, 341-347, 1994). The activated RI phosphorylatesintracellular Smad2 and Smad3, which then interact with Smad4 toregulate gene expression in the nucleus (Piek et al., FASEB J. 13,2105-2124, 1999; Massague and Chen, Genes & Development 14, 627-644,2000). Through its regulation of gene expression, TGFβ has been shown toinfluence many cellular functions such as cell proliferation, celldifferentiation, cell-cell and cell-matrix adhesion, cell motility, andactivation of lymphocytes (Massague, Ann. Rev. Cell Biol. 6, 597-641,1990; Roberts and Sporn, The transforming growth factor-betas. InPeptide growth factors and their receptors I, Sporn and Roberts, eds.(Heidelberg: Springer-Verlag), pp. 419-472, 1991). TGFβ has also beenshown or implicated in inducing or mediating the progression of manydiseases such as osteoporosis, hypertension, atherosclerosis, hepaticcirrhosis and fibrotic diseases of the kidney, liver, and lung (Blobe etal., N. Engl. J. Med. 342, 1350-1358, 2000). Perhaps, the mostextensively studied function of TGFβ is its role in tumor progression.

TGFβs have been shown to be potent growth inhibitors in various celltypes including epithelial cells (Lyons and Moses, Eur. J. Biochem. 187,467-473, 1990). The mechanism of the growth inhibition by TGFβ is mainlydue to the regulation of cell cycle-related proteins (Derynck, Trends.Biochem. Sci. 19, 548-553, 1994; Miyazono et al., Semin. Cell Biol. 5,389-398, 1994). Thus, aberrant regulation of cell cycle machinery suchas loss of retinoblastoma gene product during tumorigenesis can lead toloss of growth inhibition by TGFβ. Furthermore, mutational inactivationof TGFβ receptors, Smad2, and Smad4 has been reported in variouscarcinomas (Massague et al., Cell 103, 295-309, 2000). For example, lossof RI and/or RII expression is often observed in some humangastrointestinal cancers (Markowitz and Roberts, Cytokine, GrowthFactor, Rev. 7, 93-102, 1996).

While many carcinoma cells lose response to TGFβ's growth inhibition,they often overproduce active TGFβ isoforms when compared to theirnormal counterpart (Reiss, Microbes and Infection 1, 1327-1347, 1999).This is likely to result in the selection of cancer cells that areresistant to TGFβ's growth inhibitory activity. Indeed, an increasedlevel of TGFβ1 is strongly associated with the progression of many typesof malignancies and poor clinical outcome (Reiss, Microbes and Infection1, 1327-1347, 1999). For example, serum TGFβ1 levels have been shown tocorrelate to tumor burden, metastasis, and serum prostate specificantigen (PSA) in prostate cancer patients (Adler et al., J. Urol. 161,182-187, 1999; Shariat et al., J. Clin. Oncol. 19, 2856-2864, 2001).Consistent with these observations, marked increase of TGFβ1 and TGFβ2expression was observed in an aggressive androgen-independent humanprostate cancer cell line when compared to its less aggressiveandrogen-dependent parent cell line, LNCap (Patel et al., J. Urol. 164,1420-1425, 2000).

Several mechanisms are believed to mediate TGFβ's tumor-promotingactivity (Arteaga et al., Breast Cancer Res. Treat. 38, 49-56, 1996;Reiss, Microbes and Infection 1, 1327-1347, 1999). TGFβ is a potentimmune suppressor (Sosroseno and Herminajeng, Br. J. Biomed. Sci. 52,142-148, 1995). Overexpression of TGFβ1 in the rat prostate cancer cellswas associated with a reduced immune response during tumor formationsuggesting that TGFβ may suppress host immune response to the growingtumor (Lee et al., Prostate 39, 285-290, 1999). TGFβ has also been shownto be angiogenic in vivo (Fajardo et al., Lab. Invest. 74, 600-608,1996; Yang and Moses, J. Cell Biol. 111, 731-741, 1990; Wang et al.,Proc. Natl. Acad. Sci. U.S.A. 96, 8483-8488, 1999). Overexpression ofTGFβ during cancer progression is often associated with increasedangiogenesis and metastasis suggesting that TGFβ may promote metastasisby stimulating tumor blood vessel formation (Roberts and Wakefield,Proc. Natl. Acad. Sci. U.S.A. 100, 8621-8623, 2003). TGFβ also plays animportant role in promoting bone metastasis of human prostate and breastcancers (Koeneman et al., Prostate 39, 246-261, 1999; Yin et al., J.Clin. Invest 103, 197-206, 1999). Both TGFβ1 and TGFβ2 are produced bybone tissue, which is the largest source of TGFβ in the body (Bonewaldand Mundy, Clin. Orthop. 261-276, 1990). The latent TGFβ can beactivated by proteases such as PSA and urokinase plasminogen activator,which are abundantly secreted by cancer cells (Koeneman et al., Prostate39, 246-261, 1999). Taken together, TGFβ can act in tumormicroenvironment to promote carcinoma growth, angiogenesis, andmetastasis.

Because of its involvement in the progression of various diseases, TGFβhas been targeted for the development of novel therapeutic strategies.One way of antagonizing TGFβ activity is to utilize the ectodomain ofTGFβ type II receptor or type III receptor (betaglycan (BG)). It haspreviously been shown that ectopic expression of a soluble RIII (sBG) inhuman carcinoma cell lines can significantly inhibit tumor growth,angiogenesis, and metastasis when they are inoculated in athymic nudemice (Bandyopadhyay et al., Cancer Res. 59, 5041-5046, 1999;Bandyopadhyay et al., Oncogene 21, 3541-3551, 2002b). More recently, ithas been shown that systemic administration of recombinant sRIII caninhibit the growth, angiogenesis, and metastasis of the xenografts ofhuman breast carcinoma MDA-MB-231 cells in nude mice (Bandyopadhyay etal., Cancer Res. 62, 4690-4695, 2002a). However, the inhibition was onlypartial. This could be due, in part, to the fact that the cells producedactive TGFβ1 and active TGFβ2 and the anti-TGFβ potency of sRIII is10-fold lower for TGFβ1 than for TGFβ2 (Vilchis-Landeros et al.,Biochem. J. 355, 215-222, 2001). Interestingly, while the extracellulardomain of RII (sRII) has very low affinity for TGFβ2, its affinity forTGFβ1 and TGFβ3 is more than ten times higher than that of sRIII (Lin etal., J. Biol. Chem. 270, 2747-2754, 1995; Vilchis-Landeros et al.,Biochem. J. 355, 215-222, 2001).

While numerous TGFβ antagonists have been prepared and tested, all haveless than complete TGFβ isoform inhibiting properties. Thus, there is aneed for additional TGF antagonists or inhibitors.

SUMMARY

Certain embodiments are directed to novel heterotrimeric polypeptides inwhich the ectodomain of the TGF-β type II receptor (TβRII) is coupled tothe N- and C-terminal ends of the endoglin-domain (E domain) of theTGF-β type III receptor (TβRIII). This trimeric receptor, known as RER,can bind all three TGF-β isoforms with sub-nanomolar affinity and iseffective at neutralizing signaling induced by all three TGF-β isoforms,but not other ligands of the TGF-β superfamily, such as activins, growthand differentiation factors (GDFs), and bone morphonogenetic proteins(BMPs). The sub-nanomolar affinity of the fusion, which arises from itsability to contact the TGF-β dimer at three distinct sites, allows it toeffectively compete against the endogenous receptors for TGF-β binding.The fusion proteins described herein offer significant potential as atherapeutic agent for treating diseases driven by overexpression of theTGF-β isoforms, such as cancer and fibrosis.

Certain aspects are directed to a heterotrimeric fusion proteincomprising (a) an amino terminal segment comprising a first TGFβ bindingdomain of TGFβ receptor type II, (b) a central segment comprising aendoglin-domain of TGFβ receptor type III, and (c) a carboxy terminalsegment comprising a second TGFβ binding domain of TGFβ receptor typeII.

An example of a TGFβ type II receptor is provided as SEQ ID NO:6. Aminoacids 1 to 567 of SEQ ID NO:6 is a TGFβ receptor type-2 precursor(EC_number=2.7.11.30). The signal peptide is composed of amino acid 1 to22 of SEQ ID NO:6. The mature peptide includes amino acids 23 to 567 ofSEQ ID NO:6. The ectodomain is defined by amino acids 24 to 160 of SEQID NO:6 (RII domain). The ectodomain is followed by a transmembraneregion that spans amino acids 161 to 187 of SEQ ID NO:6. The aminoterminal segment or the carboxy terminal segment of a novelheterotrimeric fusion protein described herein can comprise,independently, an amino acid segment that is 85, 90, 95, 98, or 100%identical, including all values and ranges there between, to amino acids35, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, or 75 to145, 150, 155, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, or 170of SEQ ID NO:6, including all values and ranges there between. Thepolypeptide segment's ability to bind TGFβ can be determined by usingstandard ligand binding assays known to those of skill in the art. Incertain aspects the RII domain comprises point mutations that alter thebinding affinity of the RII domain or the binding affinity of apolypeptide comprising an RII domain. In certain aspects amino acidresidues 27, 30, 32, 50, 51, 52, 53, 55, 118, and 119 can be alteredsingly or in various combinations.

An example of a TGFβ type III receptor is provided as SEQ ID NO:7 or SEQID NO:8. Amino acids 1 to 23 of SEQ ID NO:7 or 1 to 21 of SEQ ID NO:8define the signal peptide. Amino acids 24-409 of SEQ ID NO:7 or 21-406of SEQ ID NO:8 define the endoglin-like domain (E domain or region),amino acids 410 to 783 of SEQ ID NO:7 or 407-780 of SEQ ID NO:8 definethe zona pellucida-like domain or uromodulin-like domain (U domain orregion), and amino acids 789 to 811 of SEQ ID NO:7 or 786 to 808 of SEQID NO:8 define the transmembrane region. The central segment of thetrimeric fusion protein can comprise an amino acid segment that is 85,90, 95, 98, or 100% identical, including all values and ranges therebetween, to amino acids 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 45, 50, or 60 to 350, 355, 360, 361,362, 364, 365, 370, 375, 380, 385, 390, 395, 400, 405 or 409 of SEQ IDNO:7 or SEQ ID NO:8, including all values and ranges there between. Incertain aspects the E domain comprises point mutations that alter thebinding affinity of the E domain or the binding affinity of apolypeptide comprising an E domain. In another embodiment, the centralsegment of the trimeric fusion protein can comprise an amino acidsegment that is 85, 90, 95, 98, or 100% identical, including all valuesand ranges there between, to amino acids 405, 410, 415, 420, 425, 430,440, 445, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, or 550 to500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630,640, 650, 660, 670, 690, 700, 710, 720, 730, 740, 750, 760, 770, or 780of SEQ ID NO:7 or SEQ ID NO:8, including all values and ranges therebetween. The polypeptide segment's ability to bind TGFβ can bedetermined by using standard ligand binding assays known to those ofskill in the art. In certain aspects amino acid 69, 71, 72, 90, 93, 99,108, 115, 120, 144, 163, 192, 206, 237, 252, 274, 283, and 336 of SEQ IDNO:7 can be altered singly or in various combinations, or thecorresponding amino acids of SEQ ID NO:8.

In certain aspects, the fusion protein can further comprise a linkerbetween the amino terminal segment and the central segment, and/or alinker between the central segment and the carboxy terminal segment. Ina further aspect, the linkers can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids. Incertain aspects, the amino acids of the linker are additional TGFβreceptor type II or type III amino acid sequences. In other aspects, thelinkers are not TGFβ receptor type II or type III amino acid sequences,i.e., heterologous linkers.

In certain aspects, the amino terminal segment comprises an amino acidsequence that is 85, 90, 95, 98, or 100% identical to SEQ ID NO:3,including all values and ranges there between.

In a further aspect, the central segment comprises an amino acidsequence that is 85, 90, 95, 98, or 100% identical to SEQ ID NO:4,including all values and ranges there between.

In yet a further aspect, the carboxy terminal segment comprises an aminoacid sequence that is 85, 90, 95, 98, or 100% identical to SEQ ID NO:5,including all values and ranges there between.

In certain aspects, the fusion protein has an amino acid sequence thatis 85, 90, 95, 98, or 100% identical to SEQ ID NO:2, including allvalues and ranges there between.

In a further aspect, the fusion protein can further comprise an aminoterminal signal sequence. In certain aspects, the fusion protein canfurther comprise an amino terminal or carboxy terminal tag. In certainaspects the tag is hexa-histidine.

A peptide tag as used herein refers to a peptide sequence that isattached (for instance through genetic engineering) to another peptideor a protein, to provide a function to the resultant fusion. Peptidetags are usually relatively short in comparison to a protein to whichthey are fused; by way of example, peptide tags are four or more aminoacids in length, such as, 5, 6, 7, 8, 9, 10, 15, 20, or 25 or more aminoacids. Usually a peptide tag will be no more than about 100 amino acidsin length, and may be no more than about 75, no more than about 50, nomore than about 40, or no more than about 30.

Peptide tags confer one or more different functions to a fusion protein(thereby “functionalizing” that protein), and such functions can include(but are not limited to) antibody binding (an epitope tag),purification, translocation, targeting, and differentiation (e.g., froma native protein). In addition, a recognition site for a protease, forwhich a binding antibody is known, can be used as a specificallycleavable epitope tag. The use of such a cleavable tag can provideselective cleavage and activation of a protein. Alternatively the systemdeveloped by in the Dowdy laboratory (Vocero-Akbani et al, Nat. Med.5:29-33, 1999) could be use to provide specificity of such cleavage andactivation.

Detection of the tagged molecule can be achieved using a number ofdifferent techniques. These include: immunohistochemistry,immunoprecipitation, flow cytometry, immunofluorescence microscopy,ELISA, immunoblotting (“western”), and affinity chromatography.

Epitope tags add a known epitope (antibody binding site) on the subjectprotein, to provide binding of a known and often high-affinity antibody,and thereby allowing one to specifically identify and track the taggedprotein that has been added to a living organism or to cultured cells.Examples of epitope tags include the myc, T7, GST, GFP, HA(hemagglutinin) and FLAG tags. The first four examples are epitopesderived from existing molecules. In contrast, FLAG is a syntheticepitope tag designed for high antigenicity (see, e.g., U.S. Pat. Nos.4,703,004 and 4,851,341).

Purification tags are used to permit easy purification of the taggedprotein, such as by affinity chromatography. A well-known purificationtag is the hexa-histidine (6× His) tag, literally a sequence of sixhistidine residues. The 6× His protein purification system is availablecommercially from QIAGEN (Valencia, Calif.), under the name ofQIAexpress®.

Certain embodiments are directed to the therapeutic use of the fusionsproteins described herein. Certain aspects are directed to a method oftreating a TGFβ related condition comprising administering an effectiveamount of a fusion protein described herein. The fusion protein can beadministered to a subject, such as a mammal. The mammal being treatedmay have or may be at risk for one or more conditions associated with anexcess of TGF-β for which a reduction in TGF-β levels may be desirable.Such conditions include, but are not limited to, fibrotic diseases (suchas glomerulonephritis, neural scarring, dermal scarring, pulmonaryfibrosis (e.g., idiopathic pulmonary fibrosis), lung fibrosis,radiation-induced fibrosis, hepatic fibrosis, myelofibrosis), peritonealadhesions, hyperproliferative diseases (e.g., cancer), burns,immune-mediated diseases, inflammatory diseases (including rheumatoidarthritis), transplant rejection, Dupuytren's contracture, and gastriculcers. In certain aspects the fusion protein is administerintravascularly.

Other terms related to the description provided herein include:

The term “receptor” denotes a cell-associated protein that binds to abioactive molecule (i.e., a ligand) and mediates the effect of theligand on the cell. Membrane-bound receptors are characterized by amulti-domain structure comprising an extracellular ligand-binding domainand an intracellular effector domain that is typically involved insignal transduction.

By “multimeric” or “heteromultimeric” is meant comprising two or moredifferent subunits. A “heterodimeric” receptor contains two differentsubunits, wherein a “heterotrimeric” molecule comprises three subunits.

By “soluble” multimeric receptor is meant herein a multimeric receptor,each of whose subunits comprises part or all of an extracellular domainof a receptor, but lacks part or all of any transmembrane domain, andlacks all of any intracellular domain. In general, a soluble receptor ofthe invention is soluble in an aqueous solution.

A “fusion” protein is a protein comprising two polypeptide segmentslinked by a peptide bond, produced, e.g., by recombinant processes.

As used herein, a “variant” polypeptide of a parent or wild-typepolypeptide contains one or more amino acid substitutions, deletionsand/or additions as compared to the parent or wild-type. Typically, suchvariants have a sequence identity to the parent or wild-type sequence ofat least about 90%, at least about 95%, at least about 96%, at leastabout 97%, 98%, or at least about 99%, and have preserved or improvedproperties as compared to the parent or wild-type polypeptide. Somechanges may not significantly affect the folding or activity of theprotein or polypeptide; conservative amino acid substitutions, as arewell known in the art, changing one amino acid to one having aside-chain with similar physicochemical properties (basic amino acid:arginine, lysine, and histidine; acidic amino acids: glutamic acid, andaspartic acid; polar amino acids: glutamine and asparagine; hydrophobicamino acids: leucine, isoleucine, valine; aromatic amino acids:phenylalanine, tryptophan, tyrosine; small amino acids: glycine,alanine, serine, threonine, methionine), small deletions, typically ofone to about 30 amino acids; and small amino- or carboxyl-terminalextensions, such as an amino-terminal methionine residue, a small linkerpeptide of up to about 20-25 residues, or a small extension thatfacilitates purification (an affinity tag), such as a poly-histidinetract, protein A (Nilsson et al., EMBO 1985; 14:1075 et seq.; Nilsson etal., Methods Enzymol. 1991; 198:3 et seq.), glutathione S-transferase(Smith and Johnson, Gene 1988; 67:31 et seq.), or otherantigenic:epitope or binding domain. See, in general Ford et al.,Protein Expression and Purification 1991; 2:95-107. DNAs encodingaffinity tags are available from commercial suppliers.

Sequence differences or “identity,” in the context of amino acidsequences, can be determined by any suitable technique, such as (and asone suitable selection in the context of this invention) by employing aNeedleman-Wunsch alignment analysis (see Needleman and Wunsch, J. Mol.Biol. (1970) 48:443453), such as is provided via analysis with ALIGN 2.0using the BLOSUM50 scoring matrix with an initial gap penalty of −12 andan extension penalty of −2 (see Myers and Miller, CABIOS (1989) 4:11-17for discussion of the global alignment techniques incorporated in theALIGN program). A copy of the ALIGN 2.0 program is available, e.g.,through the San Diego Super-computer (SDSC) Biology Workbench. BecauseNeedleman-Wunsch alignment provides an overall or global identitymeasurement between two sequences, it should be recognized that targetsequences which may be portions or subsequences of larger peptidesequences may be used in a manner analogous to complete sequences or,alternatively, local alignment values can be used to assessrelationships between subsequences, as determined by, e.g., aSmith-Waterman alignment (J. Mol. Biol. (1981) 147:195-197), which canbe obtained through available programs (other local alignment methodsthat may be suitable for analyzing identity include programs that applyheuristic local alignment algorithms such as FastA and BLAST programs).

The term “isolated” can refer to a nucleic acid or polypeptide that issubstantially free of cellular material, bacterial material, viralmaterial, or culture medium (when produced by recombinant DNAtechniques) of their source of origin, or chemical precursors or otherchemicals (when chemically synthesized). Moreover, an isolated compoundrefers to one that can be administered to a subject as an isolatedcompound; in other words, the compound may not simply be considered“isolated” if it is adhered to a column or embedded in an agarose gel.Moreover, an “isolated nucleic acid fragment” or “isolated peptide” is anucleic acid or protein fragment that is not naturally occurring as afragment and/or is not typically in the functional state.

Moieties of the invention, such as polypeptides or peptides may beconjugated or linked covalently or noncovalently to other moieties suchas polypeptides, proteins, peptides, supports, fluorescence moieties, orlabels. The term “conjugate” is broadly used to define the operativeassociation of one moiety with another agent and is not intended torefer solely to any type of operative association, and is particularlynot limited to chemical “conjugation.” Recombinant fusion proteins areparticularly contemplated.

The term “providing” is used according to its ordinary meaning toindicate “to supply or furnish for use.” In some embodiments, theprotein is provided directly by administering the protein, while inother embodiments, the protein is effectively provided by administeringa nucleic acid that encodes the protein. In certain aspects theinvention contemplates compositions comprising various combinations ofnucleic acid, antigens, peptides, and/or epitopes.

An effective amount means an amount of active ingredients necessary totreat, ameliorate, or mitigate a disease or a condition related to adisease. In more specific aspects, an effective amount prevents,alleviates, or ameliorates symptoms of disease, or prolongs the survivalof the subject being treated, or improves the quality of life of anindividual. Determination of the effective amount is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein. For any preparation used in themethods of the invention, an effective amount or dose can be estimatedinitially from in vitro studies, cell culture, and/or animal modelassays. For example, a dose can be formulated in animal models toachieve a desired response or circulating fusion protein concentration.Such information can be used to more accurately determine useful dosesin humans.

Other embodiments of the invention are discussed throughout thisapplication. Any embodiment discussed with respect to one aspect of theinvention applies to other aspects of the invention as well and viceversa. Each embodiment described herein is understood to be embodimentsof the invention that are applicable to all aspects of the invention. Itis contemplated that any embodiment discussed herein can be implementedwith respect to any method or composition of the invention, and viceversa. Furthermore, compositions and kits of the invention can be usedto achieve methods of the invention.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.”

Throughout this application, the term “about” is used to indicate that avalue includes the standard deviation of error for the device or methodbeing employed to determine the value.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.”

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating specific embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofthe specification embodiments presented herein.

FIG. 1. SPR sensorgrams in which increasing concentrations of the TβRIIand TβRIII_(E) were injected over a SPR sensor surface with immobilizedTGF-β2 K25R I92V K94R. The mass normalized sensorgrams are shown inpanels a and b; plots of the mass normalized equilibrium response(R_(eq)) as a function of receptor concentration ([Receptor]), alongwith fits to R_(eg)=(R_(max)×[Receptor])/(K_(d)+[Receptor]), are shownin panel c.

FIG. 2. SPR sensorgrams in which increasing concentrations of the TGF-βtype II receptor ectodomain were injected over immobilized TGF-β2 K25RI92V K94R in the absence (panel a) or presence (panel b) of a saturatingconcentration (800 nM) of the TGF-β type III receptor endoglin domain.Plots of the mass normalized equilibrium response (R_(eq)) as a functionof receptor concentration ([Receptor]), along with fits toR_(eq)=(R_(max)×[Receptor])/(K_(d)+[Receptor]), are shown in panel c.

FIG. 3. Schematic diagram of the TGFβ:TβRII complex with the TGFβ typeIII receptor endoglin domain positioned in a manner that it does notsterically overlap with either of the two bound TβRII molecules. Thelocations on the TβRII N- and C-termini are shown.

FIG. 4. SPR sensorgrams in which increasing concentrations of ER and RERwere injected over SPR surfaces with immobilized TGF-β1, -β2, and -β3.The concentrations of injected receptor range from 10 nM downward (intwo-fold increments).

FIG. 5. SPR competition binding data in which increasing concentrationsof TβRII (R), TβRIII_(E)-TβRII (ER), and TβRII-TβRIII_(E)-TβRII (RER)were pre-incubated with 0.8 nM TGF-β3 for 16 h and then injected over ahigh-density (20000 RU) SPR surface with the TGF-β monoclonal antibody1D11. Data is presented in terms of the initial slope (which is directlyproportional to the concentration of free TGF-β) as a function of thecompetitor (R, ER, or RER) concentration. Two independent measurementswere performed for each of the receptor constructs studied (designatedby the a and b suffices in the legend).

FIG. 6. Average IC₅₀ using Mv1Lu PAI1 luciferase reporter cells in96-well plates. Assays were performed using a four-fold receptor fusionand 1D11 (neutralizing antibody) dilution series and 20 pM TGF-beta 1,2, or 3 at 37° overnight.

FIGS. 7A-7C. Neutralization curves comparing various traps (RR(RII-RII), RER (RII-BG_(E)-RII), ER (BG_(E)-RII), REU(RII-BG_(E)-BG_(U), or alternatively RII-RIII), or EU (BG_(E)-BG_(U), oralternatively RIII) and 1D11 for (A) TGF-β1, (B) TGF-β2, or TGF-β3.

FIGS. 8A-8C. Neutralization curves for various RER preparations relativeto (A) TGF-β1, (B) TGF-β2, or (c) TGF-β3

DESCRIPTION

As discussed above, transforming growth factor beta (TGFβ) isoforms (β1,β2, and β3) are homodimeric polypeptides of 25 kDa. TGF-β has ninecysteine residues that are conserved among its family; eight cysteinesform four disulfide bonds within the molecule, three of which form acystine knot structure characteristic of the TGF-β superfamily, whilethe ninth cysteine forms a disulfide bond with the ninth cysteine ofanother TGF-β molecule to produce the dimer.

Though a number of TGF-β inhibitors have been reported, none have beenapproved for clinical use. The novel TGF-β inhibitor describedherein—RER—can be produced by artificially fusing together the bindingdomains of the TGFβ type II receptor and the endoglin domain of the typeIII receptor. The design of RER—a heterotrimeric fusion in which theectodomain of the TGF-β type II receptor (R) has been artificially fusedonto the N- and C-termini of the endoglin-like domain of the TGF-β typeIII receptor (E)—was conceived based on the structures of the TGF-βsbound to the their signaling receptors, TβRI and TβRII, and the resultsof surface plasmon resonance (SPR) binding studies which showed that:

1. The TGF-β type III receptor endoglin domain binds TGF-β dimers with astoichiometry of 1:1. This was shown by comparing the maximalmass-normalized SPR response as increasing concentrations of thepurified TGF-β type II receptor ectodomain (TβRII or R) and purifiedTGF-β type III receptor endoglin-like domain (TβRIII_(E) or E) wereinjected over immobilized TGF-β2 K25R I92V K94R, a variant of TGF-β2that binds TβRII with high affinity (FIGS. 1A and 1B) (De Crescenzo etal. J Mol. Biol. 355, 47-62, 2006; Baardsnes et al. Biochemistry 48,2146-55, 2009). The maximal mass-normalized response for TβRIII_(E) wasfound to be approximately one-half of that for TORII, allowing theinventors to infer that TβRIII_(E) must bind the TGF-β dimer with 1:1stoichiometry since it is well established through structural studiesthat TβRII binds TGF-β dimers with 2:1 stoichiometry (FIG. 1C) (Hart etal., Nat Struct Biol. 9, 203-8, 2002; Groppe et al., Mol Cell 29,157-68, 2008; Radaev et al., Journal of Biological Chemistry 285,14806-14, 2010).

2. TβRIII_(E) binds TGF-β dimers without displacing either of the twobound TβRIIs. This was shown by performing an SPR experiment in whichincreasing concentrations of TβRII were injected over immobilized TGF-β2K25R I92V K94R in the absence or presence of a saturating concentrationof TβRIII_(E) (800 nM) (FIGS. 2A and 2B). The data showed that themaximal mass normalized binding response for TβRII was slightlyincreased in the presence of 800 nM TβRIII_(E) (FIG. 2C), showing thatthe two receptors do not compete with one another for binding TGF-β (itis impossible for more than two TβRIIs to bind the TGF-β dimer, and thusthe increase in the maximal amplitude is likely caused by anexperimental artifact, such as a mismatch in the concentrations ofTβRIII_(E) in the TβRII samples that were injected and the buffer).

Together, these observations suggest that TGF-β dimers are capable offorming a heterotrimeric complex in which each TGF-β dimer binds twomolecules of TβRII and one molecule of TβRIII_(E). The structure of theTGF-β bound to TβRII has been reported (Hart et al., Nat Struct Biol. 9,203-8, 2002; Groppe et al., Mol Cell 29, 157-68, 2008; Radaev et al.,Journal of Biological Chemistry 285, 14806-14, 2010), but the structureof TβRIII_(E), either alone or bound to TGF-β, has not. This has led tothe hybrid structure where the precise structure of TβRIII_(E) is notknown, but its overall positioning between the two bound TβRIIs on thedistal ends of the TGF-β dimer is known (FIG. 3).

This hybrid model for binding of TβRII and TβRIII_(E) led to theconstruction of the heterotrimeric RER (TβRII-TβRIII_(E)-TβRII) fusionas a novel inhibitor for binding and sequestering TGF-β. The inclusionof an additional binding domain enhanced the affinity of the fusion forthe TGF-βs, especially TGF-β1 and TGF-β3, which bind TβRII with high(K_(d) ˜120 nM) affinity (Baardsnes et al. Biochemistry 48, 2146-55,2009; Radaev et al., Journal of Biological Chemistry 285, 14806-14,2010).

In comparison to the currently described RER, Genzyme's monoclonalantibody GC1008 (the humanized version of the mouse monoclonal antibody1D11) has been shown to bind the three TGF-β isoforms with a K_(d) ofapproximately 5-10 nM (Grütter, et. al., PNAS U.S.A. 105(51): 20251-56,2008), but it has not proven to be very effective in clinical trials formalignant melanoma and renal cell carcinoma. The reason for the lack ofeffectiveness might be that GC1008 does not bind the TGF-βs tightlyenough to compete against the cell surface TGF-β receptors, which bindthe TGF-βs at picomolar to sub-picomolar concentrations.

The polypeptides described herein include high affinity heterotrimericTGF-β inhibitors, such as RER. As described above RER has been shown tobind all three TGF-β isoforms with low nanomolar affinity tosub-nanomolar affinity. RER is more potent than the monoclonal antibody1D11. Thus, owing to its enhanced affinity for binding TGF-β, RER moreeffectively competes against the cell surface receptors for bindingTGF-β, and in turn blocking its disease-promoting properties in cancerand fibrosis for example.

An example of an RER amino acid sequence (for example see SEQ ID NO:2)has one or more of the following features:

1. In certain aspects the TβRII sequence is human (SEQ ID NO:6), whilethe TβRIII_(E) sequence can be rat (SEQ ID NO:7). In certain aspects theTβRIII_(E) sequence can be human (SEQ ID NO:8).

2. In certain embodiments the N-terminal TβRII sequence of RER extendsfrom residue 42-160 of SEQ ID NO:6, while the C-terminal TβRII sequenceof RER extends from residue 48-160 of SEQ ID NO:6.

3. In certain embodiments the TβRIII_(E) sequence extends from residue24-383 of SEQ ID NO:7. In certain aspects, the TβRIII_(E) sequenceincludes 1, 2, 3, and/or 4 single amino acid substitutions relative tothe wild type rat sequence (SEQ ID NO:7), R58H, H116R, C278S, and N337A.

4. In certain embodiments there is no linker between TβRIII_(E) and theC-terminal TβRII domain. In other aspects a Lys-Leu dipeptide encoded bythe HindIII restriction site used to join the corresponding DNAfragments together forms a linker. It is contemplated that any dipeptidecan be used.

5. In certain embodiments there is an 18 amino acid linker with thesequenceGly-Leu-Gly-Pro-Val-Glu-Ser-Ser-Pro-Gly-His-Gly-Leu-Asp-Thr-Ala-Ala-Ala(SEQ ID NO:9) that links the C-terminus of the N-terminal TβRII to theN-terminus of TβRIII_(E).

6. In certain embodiments there is a C-terminal hexa-histidine tag (forpurification purposes).

In one example, an RER expression cassette was inserted downstream ofthe albumin signal peptide and an engineered NotI cloning site with thesequenceMet-Lys-Trp-Val-Thr-Phe-Leu-Leu-Leu-Leu-Phe-Ile-Ser-Gly-Ser-Ala-Phe-Ser-Ala-Ala-Ala(SEQ ID NO:10). The entire albumin signal peptide was placed downstreamof the CMV promoter in a modified form of pcDNA3.1 (Invitrogen) aspreviously described (Zou and Sun, Cell 134, 215-30, 2004).

A plasmid expressing RER construct was transfected into CHO Lec 3.2.8.1cells (Rosenwald et al., Mol Cell Biol. 9(3):914-24, 1989) and stabletransfectants were selected using MSX (Zou and Sun, Cell 134, 215-30,2004). The stable transfectants were in turn screened for high levelexpression of the RER fusion by examining the conditioned medium using apolyclonal antibody raised against the rat betaglycan ectodomain. Theclone expressing RER at the highest level was expanded and ultimatelytransferred into serum free medium for production of conditioned medium.The RER was then purified from the conditioned medium by passing it overa NiNTA column, washing it with 25 mM Tris, 100 mM NaCl, and 10 mMimidazole, pH 8 and ultimately by eluting it with the same buffer, butwith 300 mM imidazole.

The isolated RER fusion protein was in turn characterized by performingan SPR experiment in which it, together with similarly prepared ER (i.e.the previously described TβRIII_(E)TβRII fusion (Verona et al., ProteinEng Des Sel. 21, 463-73, 2008), except produced in CHO cells, notbacteria), was injected over a SPR sensor chip with immobilized TGF-β1,-β2, and -β3. This data showed comparable on-rates, but significantlyslower off-rates, especially for TGF-β1 and TGF-β3 (FIG. 4). Thisqualitatively shows that RER binds the TGF-βs with higher affinity thanER; however, the magnitude of this increase proved to be difficult toquantify since the slow association precluded accurate measurement ofthe equilibrium SPR response, especially at lower concentrations ofinjected receptor.

To further evaluate affinity, an SPR competition experiment wasperformed in which the commercially available TGF-β monoclonal antibody1D11 (R&D Systems) was coupled to an SPR sensor chip at high density(20000 RU) and in turn increasing concentration of R (TβRII), ER(BG_(E)-RII), or RER(RII-BG_(E)-RII) were injected in the presence of afixed low (0.8 nM) concentration of TGF-β3. The initial slope of thesesensorgrams (which is a linear function of the free TGF-β3concentration) was then plotted as a function of the concentration ofthe receptor fusion (FIG. 5). This showed that RER is indeed a morepotent competitor than ER, consistent with the slower dissociation ratefor RER compared to ER.

RER polypeptides demonstrate more potent activity relative to similarfusion proteins. For example the average IC₅₀ [nM] using Mv1Lu PAI1luciferase reporter cells in 96-well plates is markedly lower for RERpolypeptides (FIG. 6). Neutralization curves comparing various receptorfusions (RR (RII-RII, also known as T22d35), RER (RII-BG_(E)-RII), ER(BG_(E)-RII), REU (RII-BG_(E)-BG_(U) or alternatively RII-RIII), or EU(BG_(E)-BG_(U) or alternatively RIII) and 1D11 for (A) TGF-β1, (B)TGF-β2, or TGF-β3 also show an improved activity for RER polypeptides(FIG. 7 and FIG. 8).

I. Linkers

In some embodiments, the invention provides a fusion protein comprisingthree TGF-β binding domains joined to each other directly or by alinker, such as, e.g., a short peptide linker. In some embodiments, theC terminus of the amino terminal TGF-β binding segment is joined by apeptide linker to the N terminus of the central TGF-β binding segment,and the C terminus of the center TGFβ binding segment may be joined tothe N terminus of the carboxy TGFβ binding segment by a second linker. Alinker is considered short if it contains 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, to 50 or fewer amino acids.

Most typically, the linker is a peptide linker that contains 50 or feweramino acids, e.g., 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 3, 4,2, or 1 amino acid(s). In certain aspects, the sequence of the peptidelinker is a non-TGF-β type II or type III receptor amino acid sequence.In other aspects, the sequence of the peptide linker is additional TGF-βtype II or type III receptor amino acid sequence, e.g., the 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, to 50 orfewer amino acids flanking the carboxy an/or amino terminal ends of thebinding domains. The term additional in this context refers to aminoacids in addition to those that define the segments of theheterotrimeric polypeptide as defined above. In various embodiments, thelinker does not contain more than 50, 40, 20, 10, or 5 contiguous aminoacids from the native receptor sequences. Typically, the linker will beflexible and allow the proper folding of the joined domains. Amino acidsthat do not have bulky side groups and charged groups are generallypreferred (e.g., glycine, serine, alanine, and threonine). Optionally,the linker may additionally contain one or more adaptor amino acids,such as, for example, those produced as a result of the insertion ofrestriction sites. Generally, there will be no more than 10, 9, 8, 7, 6,5, 4, 3, 2 adaptor amino acids in a linker.

In some embodiments, the linker comprises one or more glycines, e.g., 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 18, or more glycines. Forexample, the linker may consist of (GGG)n, where n=1, 2, 3, 4, 5, 6, 7,etc. and optional adaptor amino acids. In certain aspects, the linker isa glycine-serine linker which comprises (GGGS)n, where n=1, 2, 3, 4, 5,etc. In view of the results disclosed herein, the skilled artisan willrecognize that any other suitable peptide linker can be used in thefusion proteins of the invention, for example, as described in Alfthanet al., Protein Eng. 8:725-31, 1995; Argos, J. MoI. Biol. 211:943-58,1990; Crasto et al., Protein Eng., 13:309-12, 2000; Robinson et al.,PNAS USA, 95:5929-34, 1998.

II. Nucleic Acids, Vectors, Host Cells

The invention further provides nucleic acids encoding any of the fusionproteins of the invention, vectors comprising such nucleic acids, andhost cells comprising such nucleic acids. For example, in anillustrative embodiment, the nucleic acid of the invention comprises thesequence as set forth in SEQ ID NO:1.

Nucleic acids of the invention can be incorporated into a vector, e.g.,an expression vector, using standard techniques. The expression vectormay then be introduced into host cells using a variety of standardtechniques such as liposome-mediated transfection, calcium phosphateprecipitation, or electroporation. The host cells according to thepresent invention can be mammalian cells, for example, Chinese hamsterovary cells, human embryonic kidney cells (e.g., HEK 293), HeLa S3cells, murine embryonic cells, or NSO cells. However, non-mammaliancells can also be used, including, e.g., bacteria, yeast, insect, andplant cells. Suitable host cells may also reside in vivo or be implantedin vivo, in which case the nucleic acids could be used in the context ofin vivo or ex vivo gene therapy.

III. Methods of Making

The invention also provides methods of producing (a) fusion proteins,(b) nucleic acid encoding the same, and (c) host cells andpharmaceutical compositions comprising either the fusion proteins ornucleic acids. For example, a method of producing the fusion proteinaccording to the invention comprises culturing a host cell, containing anucleic acid that encodes the fusion protein of the invention underconditions resulting in the expression of the fusion protein andsubsequent recovery of the fusion protein. In one aspect, the fusionprotein is expressed in CHO or HEK 293 cells and purified from themedium using methods known in the art. In some embodiments, the fusionprotein is eluted from a column at a neutral pH or above, e.g., pH 7.5or above, pH 8.0 or above, pH 8.5 or above, or pH 9.0 or above.

The fusion proteins, including variants, as well as nucleic acidsencoding the same, can be made using any suitable method, includingstandard molecular biology techniques and synthetic methods, forexample, as described in the following references: Maniatis (1990)Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., and Bodansky et al. (1995) ThePractice of Peptide Synthesis, 2nd ed., Spring Verlag, Berlin, Germany).Pharmaceutical compositions can also be made using any suitable method,including for example, as described in Remington: The Science andPractice of Pharmacy, eds. Gennado et al., 21th ed., Lippincott,Williams & Wilkins, 2005).

IV. Pharmaceutical Compositions and Methods of Administration

The invention provides pharmaceutical compositions comprising the fusionproteins of the invention or nucleic acids encoding the fusion proteins.

The fusion protein may be delivered to a cell or organism by means ofgene therapy, wherein a nucleic acid sequence encoding the fusionprotein is inserted into an expression vector that is administered invivo or to cells ex vivo, which are then administered in vivo, and thefusion protein is expressed therefrom. Methods for gene therapy todeliver TGF-β antagonists are known (see, e.g., Fakhrai et al., PNASUSA, 93:2909-14, 1996 and U.S. Pat. No. 5,824,655).

The fusion protein may be administered to a cell or organism in apharmaceutical composition that comprises the fusion protein as anactive ingredient. Pharmaceutical compositions can be formulateddepending upon the treatment being effected and the route ofadministration. For example, pharmaceutical compositions of theinvention can be administered orally, topically, transdermally,parenterally, subcutaneously, intravenously, intramuscularly,intraperitoneally, by intranasal instillation, by intracavitary orintravesical instillation, intraocularly, intraarterially,intralesionally, or by application to mucous membranes, such as, that ofthe nose, throat, and bronchial tubes. The pharmaceutical compositionwill typically comprise biologically inactive components, such asdiluents, excipients, salts, buffers, preservants, etc. Standardpharmaceutical formulation techniques and excipients are well known topersons skilled in the art (see, e.g., Physicians' Desk Reference (PDR)2005, 59th ed., Medical Economics Company, 2004; and Remington: TheScience and Practice of Pharmacy, eds. Gennado et al. 21th ed.,Lippincott, Williams & Wilkins, 2005).

Generally, the fusion protein of the invention may be administered as adose of approximately from 1 μg/kg to 25 mg/kg, depending on theseverity of the symptoms and the progression of the disease. Theappropriate therapeutically effective dose of an antagonist is selectedby a treating clinician and would range approximately from 1 μg/kg to 20mg/kg, from 1 μg/kg to 10 mg/kg, from 1 μg/kg to 1 mg/kg, from 10 μg/kgto 1 mg/kg, from 10 μg/kg to 100 μg/kg, from 100 μg to 1 mg/kg, and from500 μg/kg to 5 mg/kg. Effective dosages achieved in one animal may beconverted for use in another animal, including human, using conversionfactors known in the art (see, e.g., Freireich et al., Cancer Chemother.Reports, 50(4):219-244 (1996)).

V. Therapeutic and Non-Therapeutic Uses

The fusion proteins of the invention may be used to capture orneutralize TGF-β, thus reducing or preventing TGF-β binding to naturallyoccurring TGF-β receptors.

The invention includes a method of treating a subject (e.g., mammal) byadministering to the mammal a fusion protein described herein or anucleic acid encoding the fusion protein or cells containing a nucleicacid encoding the fusion protein. The mammal can be for example, primate(e.g., human), rodent (e.g., mouse, guinea pig, rat), or others (suchas, e.g., dog, pig, rabbit).

The mammal being treated may have or may be at risk for one or moreconditions associated with an excess of TGF-β for which a reduction inTGF-β levels may be desirable. Such conditions include, but are notlimited to, fibrotic diseases (such as glomerulonephritis, neuralscarring, dermal scarring, pulmonary fibrosis (e.g., idiopathicpulmonary fibrosis), lung fibrosis, radiation-induced fibrosis, hepaticfibrosis, myelofibrosis), peritoneal adhesions, hyperproliferativediseases (e.g., cancer), burns, immune-mediated diseases, inflammatorydiseases (including rheumatoid arthritis), transplant rejection,Dupuytren's contracture, and gastric ulcers.

In certain embodiments, the fusion proteins, nucleic acids, and cells ofthe invention are used to treat diseases and conditions associated withthe deposition of extracellular matrix (ECM). Such diseases andconditions include, but are not limited to, systemic sclerosis,postoperative adhesions, keloid and hypertrophic scarring, proliferativevitreoretinopathy, glaucoma drainage surgery, corneal injury, cataract,Peyronie's disease, adult respiratory distress syndrome, cirrhosis ofthe liver, post myocardial infarction scarring, restenosis (e.g.,post-angioplasty restenosis), scarring after subarachnoid hemorrahage,multiple sclerosis, fibrosis after laminectomy, fibrosis after tendonand other repairs, scarring due to tatoo removal, biliary cirrhosis(including sclerosing cholangitis), pericarditis, pleurisy,tracheostomy, penetrating CNS injury, eosinophilic myalgic syndrome,vascular restenosis, veno-occlusive disease, pancreatitis and psoriaticarthropathy. In particular, the fusion proteins, and related aspects ofthe invention are particularly useful for the treatment of peritonealfibrosis/adhesions. It is well known that antibodies are readilytransferred from the peritoneal cavity into circulation. Therefore,intraperitoneal delivery of the fusion protein may provide a highlylocalized form of treatment for peritoneal disorders like peritonealfibrosis and adhesions due to the advantageous concentration of thefusion protein within the affected peritoneum.

The fusion proteins, nucleic acids, and cells of the invention are alsouseful to treat conditions where promotion of re-epithelialization isbeneficial. Such conditions include, but are not limited to: diseases ofthe skin, such as venous ulcers, ischaemic ulcers (pressure sores),diabetic ulcers, graft sites, graft donor sites, abrasions and burns;diseases of the bronchial epithelium, such as asthma and ARDS; diseasesof the intestinal epithelium, such as mucositis associated withcytotoxic treatment, esophagial ulcers (reflex disease), stomach ulcers,and small intestinal and large intestinal lesions (inflammatory boweldisease).

Still further uses of the fusion proteins, nucleic acids, and cells ofthe invention are in conditions in which endothelial cell proliferationis desirable, for example, in stabilizing atherosclerotic plaques,promoting healing of vascular anastomoses, or in conditions in whichinhibition of smooth muscle cell proliferation is desirable, such as inarterial disease, restenosis and asthma.

The fusion proteins, nucleic acids, and cells of the invention are alsouseful in the treatment of hyperproliferative diseases, such as cancersincluding, but not limited to, breast, prostate, ovarian, stomach, renal(e.g., renal cell carcinoma), pancreatic, colorectal, skin, lung,thyroid, cervical and bladder cancers, glioma, glioblastoma,mesothelioma, melanoma, as well as various leukemias and sarcomas, suchas Kaposi's Sarcoma, and in particular are useful to treat or preventrecurrences or metastases of such tumors. In particular embodiments, thefusion proteins, nucleic acids, and cells of the invention are useful inmethods of inhibiting cyclosporin-mediated metastases. It will of coursebe appreciated that in the context of cancer therapy, “treatment”includes any medical intervention resulting in the slowing of tumorgrowth or reduction in tumor metastases, as well as partial remission ofthe cancer in order to prolong life expectancy of a patient. In oneembodiment, the invention is a method of treating cancer comprisingadministering a fusion protein, nucleic acid or cells of the invention.In particular embodiments, the condition is renal cancer, prostatecancer or melanoma.

The fusion proteins, nucleic acids, and cells of the invention are alsouseful for treating, preventing and reducing the risk of occurrence ofrenal insufficiencies including, but not limited to, diabetic (type Iand type II) nephropathy, radiational nephropathy, obstructivenephropathy, diffuse systemic sclerosis, pulmonary fibrosis, allograftrejection, hereditary renal disease (e.g., polycystic kidney disease,medullary sponge kidney, horseshoe kidney), nephritis,glomerulonephritis, nephrosclerosis, nephrocalcinosis, systemic lupuserythematosus, Sjogren's syndrome, Berger's disease, systemic orglomerular hypertension, tubulointerstitial nephropathy, renal tubularacidosis, renal tuberculosis, and renal infarction. In particularembodiments, the fusion proteins, nucleic acids and cells of theinvention are combined with antagonists of therenin-angiotensin-aldosterone system including, but not limited to,renin inhibitors, angiotensin-converting enzyme (ACE) inhibitors, Ang Iireceptor antagonists (also known as “Ang Il receptor blockers”), andaldosterone antagonists (see, for example, WO 2004/098637).

The fusion proteins, nucleic acids, and cells of the invention are alsouseful to enhance the immune response to macrophage-mediated infections,such as those caused by Leishmania spp., Trypanosoma cruzi,Mycobacterium tuberculosis and Mycobacterium leprae, as well as theprotozoan Toxoplasma gondii, the fungi Histoplasma capsulatum, Candidaalbicans, Candida parapsilosis, and Cryptococcus neoformans, andRickettsia, for example, R. prowazekii, R. coronii, and R.tsutsugamushi. They are also useful to reduce immunosuppression caused,for example, by tumors, AIDS or granulomatous diseases.

In addition, without being bound to any particular theory, it is alsobelieved that the fusion proteins of the invention, because they lack animmunoglobulin domain (unlike TGF-β antibodies and TGF-β receptor-Fcfusion proteins) may not be as susceptible to clearance from sites ofaction by the immune system (e.g., in conditions or diseases of thelung).

The invention claimed is:
 1. A TGFβ-binding heterotrimeric fusionprotein wherein the fusion protein has an amino acid sequence that is90% identical to SEQ ID NO:
 2. 2. The fusion protein of claim 1, furthercomprising an amino terminal signal sequence.
 3. The fusion protein ofclaim 1, further comprising an amino terminal or carboxy terminal tag.4. The fusion protein of claim 3, wherein the tag is a carboxy terminalhexa-histidine.
 5. A method of treating a condition related to increasedexpression TGFβ comprising administering an effective amount of thefusion protein of claim 1 to subject in thereof.
 6. The method of claim5, wherein the condition is a hyperproliferative disorder.
 7. The methodof claim 6, wherein the hyperproliferative disorder is cancer.
 8. Themethod of claim 5, wherein the condition is fibrosis.
 9. Aheterotrimeric fusion protein wherein the fusion protein has the aminoacid sequence of SEQ ID NO:2.
 10. The fusion protein of claim 9, furthercomprising an amino terminal signal sequence.
 11. The fusion protein ofclaim 9, further comprising an amino terminal or carboxy terminal tag.12. The fusion protein of claim 11, wherein the tag is a carboxyterminal hexa-Histidine.
 13. A fusion protein comprising, in theN-terminal to C-terminal direction: (i) a first portion having an aminoacid sequence that is at least 90% identical to the amino acid sequenceof SEQ ID NO: 3; (ii) a second portion having an amino acid sequencethat is at least 90% identical to the amino acid sequence of SEQ ID NO:4; and (iii) a third portion having an amino acid sequence that is atleast 90% identical to the amino acid sequence of SEQ ID NO:
 5. 14. Thefusion protein of claim 13, wherein the first portion has an amino acidsequence that is at least 95% identical to the amino acid sequence ofSEQ ID NO:
 3. 15. The fusion protein of claim 13, wherein the secondportion has an amino acid sequence that is at least 95% identical to theamino acid sequence of SEQ ID NO:
 4. 16. The fusion protein of claim 13,wherein the third portion has an amino acid sequence that is at least95% identical to the amino acid sequence of SEQ ID NO:
 5. 17. The fusionprotein of claim 13, wherein the first portion has an amino acidsequence that is at least 95% identical to the amino acid sequence ofSEQ ID NO: 3, the second portion has an amino acid sequence that is atleast 95% identical to the amino acid sequence of SEQ ID NO: 4, and thethird portion has an amino acid sequence that is at least 95% identicalto the amino acid sequence of SEQ ID NO:
 5. 18. The fusion protein ofclaim 13, wherein the first portion has the amino acid sequence of SEQID NO:
 3. 19. The fusion protein of claim 13, wherein the second portionhas the amino acid sequence of SEQ ID NO:
 4. 20. The fusion protein ofclaim 13, wherein the third portion has the amino acid sequence of SEQID NO:
 5. 21. The fusion protein of claim 13, wherein the first portionhas the amino acid sequence of SEQ ID NO: 3, the second portion has theamino acid sequence of SEQ ID NO: 4, and the third portion has the aminoacid sequence of SEQ ID NO:
 5. 22. The fusion protein of claim 13,wherein either or both of (i) the first portion and the second portion,and (ii) the second portion and the third portion are joined to oneanother by way of a polypeptide linker.
 23. The fusion protein of claim13, further comprising an amino terminal or carboxy terminal tag.
 24. Anucleic acid encoding the fusion protein of claim
 13. 25. The nucleicacid of claim 24, wherein the first portion has an amino acid sequencethat is at least 95% identical to the amino acid sequence of SEQ ID NO:3.
 26. The nucleic acid of claim 24, wherein the second portion has anamino acid sequence that is at least 95% identical to the amino acidsequence of SEQ ID NO:
 4. 27. The nucleic acid of claim 24, wherein thethird portion has an amino acid sequence that is at least 95% identicalto the amino acid sequence of SEQ ID NO:
 5. 28. The nucleic acid ofclaim 24, wherein the first portion has an amino acid sequence that isat least 95% identical to the amino acid sequence of SEQ ID NO: 3, thesecond portion has an amino acid sequence that is at least 95% identicalto the amino acid sequence of SEQ ID NO: 4, and the third portion has anamino acid sequence that is at least 95% identical to the amino acidsequence of SEQ ID NO:
 5. 29. The nucleic acid of claim 24, wherein thefirst portion has the amino acid sequence of SEQ ID NO:
 3. 30. Thenucleic acid of claim 24, wherein the second portion has the amino acidsequence of SEQ ID NO:
 4. 31. The nucleic acid of claim 24, wherein thethird portion has the amino acid sequence of SEQ ID NO:
 5. 32. Thenucleic acid of claim 24, wherein the first portion has the amino acidsequence of SEQ ID NO: 3, the second portion has the amino acid sequenceof SEQ ID NO: 4, and the third portion has the amino acid sequence ofSEQ ID NO:
 5. 33. The nucleic acid of claim 24, wherein either or bothof (i) the first portion and the second portion, and (ii) the secondportion and the third portion are joined to one another by way of apolypeptide linker.
 34. The nucleic acid of claim 24, wherein the fusionprotein further comprises an amino terminal or carboxy terminal tag. 35.A vector comprising the nucleic acid of claim
 24. 36. A host cellcomprising the nucleic acid of claim
 24. 37. A host cell comprising thevector of claim
 35. 38. A method of producing the fusion protein ofclaim 13, the method comprising contacting a host cell with a nucleicacid encoding the fusion protein and subsequently isolating the fusionprotein from the host cell.
 39. A pharmaceutical composition comprisingthe fusion protein of claim 13 and one or more pharmaceuticallyacceptable excipients.
 40. The pharmaceutical composition of claim 39,wherein the composition is formulated for administration to a humansubject.
 41. The pharmaceutical composition of claim 40, wherein thecomposition is formulated for parenteral, subcutaneous, intravenous,intramuscular, or intraperitoneal administration to the human subject.42. A method of reducing or preventing binding of TGF-β to one or moreendogenous TGF-β receptors in a subject, the method comprisingadministering to the subject the fusion protein of claim
 13. 43. Themethod of claim 42, wherein the subject is a human.